Cytotoxic Lymphocyte Precursors Research Articles

ARE THE FUNCTIONAL ASSAYS USEFUL IN PREDICTING GRAFT-VERSUS-HOST DISEASE IN HLA-MATCHED SIBLING BONE MARROW TRANSPLANTATION?

A reply to the letter from Affaticati et al. There is no doubt that the frequency analysis of helper T lymphocyte precursors (HTLp) and cytotoxic T lymphocyte precursors (CTLp) has great impact on the detection of alloreactivity. However, the debate on the clinical values of these functional assays for the prediction of graft-versus-host disease (GVHD) occurrence after bone marrow transplantation (BMT) has carried on for the last decade. One can easily find several reports in the literature either for (1–4) or against (5–9) the usefulness of these functional assays for predicting GVHD in HLA-matched sibling BMT. Although our observation (9) showed no significant correlation between the HTLp/CTLp frequencies and the incidence of acute GVHD after HLA-matched sibling BMT, there are other reports showing the contrary (5,7). Due to the lack of standard protocols for these functional assays, there are considerable variations in the methodology used by different investigators, which makes it difficult, sometimes impossible, to compare or correlate data from different research groups. For a rigorous examination on the association between the frequencies of HTLp/CTLp and the incidence of GVHD, it is crucial to establish standard protocols and to study a sufficient number of transplant recipients with homogeneity of disease and uniformity of conditioning and GVHD prophylaxis. The effectiveness of the immunosuppressive regimen after transplantation and the precision of the HLA matching also need to be taken into consideration. The discordance in one or more of these factors could be attributable to the controversy regarding the usefulness of HTLp/CTLp frequency estimation for GVHD prediction. During the last few years, the sensitivity of these assays has been improved by eliminating antigen-presenting cells from the responder population and/or T cells from the stimulator population (10,11). However, the complexity of the assay procedure makes these protocols impracticable for routine clinical use, and researchers are still searching for a better method. The combined limiting dilution assay (LDA) used in our study was established by Bouma (12) et al. and has proven to be as sensitive as single LDA to detect alloreactive HTLp and CTLp frequencies in HLA-mismatched settings. Our observation (9) has demonstrated that this combined LDA may not be ideal in HLA-identical sibling settings. The possible reasons have been discussed in detail in our previous articles (8,9). Although Affaticati et al. (13), using a single LDA, showed a positive correlation of CTLp frequency with clinical acute GVHD grades II–IV, the statistical significance remained marginal (P =0.04), with a third of patients who did not develop severe GVHD demonstrating high frequencies. It may well be that the positive CTLp frequencies detected in both publications by Affaticati et al. and ourselves were against minor histocompatibility antigens. Some of these responses detected in cohorts of patients developing no or low-grade GVHD may, however, not be relevant or strong enough in the in vivo clinical setting (in the presence of prophylaxis regimens for example) to give rise to GVHD. However, in our view, CTLp frequency estimation remains an inconclusive predictive indicator of GVHD occurrence and severity in the HLA-matched sibling setting, mainly due to overlapping results of high frequency within cohorts of patients demonstrating either no or low-grade GVHD (grades 0–1). Anne Dickinson Xiao-Nong Wang

Immunotherapy of melanoma.

Over the past decade, tumour immunology has progressed enormously, as a result of the application of a range of new technologies. Screening of tumour gene expression libraries using cancer patients' sera and cancer-specific T lymphocytes has defined a large number of tumour-specific antigenic proteins. The repertoire of known tumour antigens is rapidly expanding as a consequence of these advances. In addition, over the last few years the analysis of human T-cell responses specific for several tumour antigens in cancer patients has been made possible by the use of novel staining reagents (i.e. tetramers). This has allowed the characterization and monitoring of specific tumour responses, and has provided an opportunity to greatly accelerate the development of new anticancer vaccines. Recent results have demonstrated that the immune system can detect the presence of malignant cells in cancer patients, and have raised hopes for the use of antigenic cancer proteins as vaccines to induce tumour-specific cell-mediated immunity. Tumour immunology is now on solid technical and conceptual footing. However, the challenge remains to apply to patients the results obtained in the laboratories.

HLA-C mismatches induce strong cytotoxic T-cell reactivity in the presence of an additional DRB/DQB mismatch and affect NK cell-mediated alloreactivity.

The functional relevance of HLA-C mismatches in an alloresponse is still much debated, putting into doubt the relevance of matching for this antigen in selection of an allogeneic bone marrow donor. In addition to presenting peptides to T cells, HLA-C also functions as a ligand for killing inhibitory receptors (KIRs) on natural killer (NK) cells. In the current study we provide an in vitro basis to address the question of whether mismatches for this antigen are a risk factor for acute graft-versus-host disease (GVHD). By analysis of cytotoxic and helper T-lymphocyte precursor frequency (CTLp-f and HTLp-f) in 153 pairs, we are able to show that isolated HLA-C mismatches appear less immunogenic than do isolated HLA-A mismatches. Strikingly, the presence of an HLA-C mismatch next to a HLA-DRB or HLA-DQB mismatch leads to a synergistic increase in CTLp-f outcome. Moreover, we are the first to show that absence of a single inhibitory epitope as a result of an HLA-C mismatch can be sufficient to induce NK mediated alloreactivity, that is, kill and proliferate. We conclude that, in most cases, isolated HLA-C mismatches may be acceptable with respect to T-cell-mediated alloreactivity; however, the presence of a strong helper epitope (DR/DQ mismatch) appears sufficient to overcome the low immunogenicity of HLA-C. HLA-C mismatches that affect KIR epitopes, can induce NK mediated alloreactivity. This suggests that, in HLA-A-, -B-, -DR-, and -DQ-matched patients, NK cells may play a role in the induction and development of acute GVHD.

Low level viral persistence after infection with LCMV: a quantitative insight through numerical bifurcation analysis

Many important viruses persist at very low levels in the body in the face of host immunity, and may influence the maintenance of this state of `infection immunity'. To analyse low level viral persistence in quantitative terms, we use a mathematical model of antiviral cytotoxic T lymphocyte (CTL) response to lymphocytic choriomeningitis virus (LCMV).This model, described by a non-linear system of delay differential equations (DDEs), is studied using numerical bifurcation analysis techniques for DDEs. Domains where low level LCMV coexistence with CTL memory is possible, either as an equilibrium state or an oscillatory pattern, are identified in spaces of the model parameters characterising the interaction between virus and CTL populations. Our analysis suggests that the coexistence of replication competent virus below the conventional detection limit (of about 100 pfu per spleen) in the immune host as an equilibrium state requires the per day relative growth rate of the virus population to decrease at least 5-fold compared to the acute phase of infection. Oscillatory patterns in the dynamics of persisting LCMV and CTL memory, with virus population varying between 1 and 100 pfu per spleen, are possible within quite narrow intervals of the rates of virus growth and precursor CTL population death. Whereas the virus replication rate appears to determine the stability of the low level virus persistence, it does not affect the steady-state level of the viral population, except for very low values.

Peptide analogues of a subdominant epitope expressed in ebv-associated tumors: synthesis and immunological activity.

H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide is an EBV subdominant epitope that represents the target of HLA-A2 restricted CTL responses. The CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses. In contrast, the [Tyr(1), Ala(3)]CLG (YLA) analogue showed high affinity for HLA-A2 molecules and efficiently stimulated CLG-specific CTL precursors. Nevertheless, this modified epitope showed low enzymatic stability. To further improve the immunotherapeutical potential of this "improved epitope", we have synthesized and tested YLA analogues containing different modifications next to the scissile peptide bond. Among the analogues we found three peptides, with higher enzymatic resistance, that efficiently stimulate CTL responses. These peptides may be used for EBV-specific immunotherapies.

Permissible and immunogenic HLA-A mismatches: cytotoxic T-cell precursor frequencies reflect graft survival data

Analysis of the in vivo immunogenicity of single HLA mismatches, in the context of a patient’s own human leukocyte antigen (HLA) phenotype, has been used to define permissible and immunogenic HLA mismatches. Kidney graft survival in the case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. The present study tested whether such permissible and immunogenic HLA mismatches are reflected in the in vitro cytotoxic T-lymphocyte (CTL) allorepertoire. Limiting dilution experiments were performed to analyze the number of precursor CTL directed against individual HLA class I antigens. In general, the frequency of CTLp directed against permissible HLA-A antigens ( n = 70, mean frequency 27 CTLp per million peripheral blood lymphocytes [PBL]) was found to be significantly lower compared with the CTLp directed against immunogenic HLA-A antigens ( n = 73, mean frequency 59 CTLp per million PBL). The difference was found both in healthy individuals and a population of renal transplant candidates. These results were confirmed by a retrospective analysis of CTLp frequencies performed between partly mismatched unrelated bone marrow donors and their potential recipients. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a particular patient should be determined on an individual basis.

High anti-paternal cytotoxic T-lymphocyte precursor frequencies in women with unexplained recurrent spontaneous abortions.

A number of cases of unexplained (idiopathic) recurrent spontaneous abortions may be attributable to immunological mechanisms. Several lines of evidence indicate that some immunocompetent effector cell populations play an important role in the pathogenesis of unexplained miscarriages. However a suitable method is lacking for defining an existing immunological background of recurrent spontaneous abortions. We tried to find a useful cellular immunological method, that is suitable for predicting the eventual immunological cause in the case of unexplained recurrent spontaneous abortions. We have examined the anti-paternal cytotoxic T-lymphocyte precursor frequencies by cell-mediated lympholysis and limiting dilution analysis in the peripheral blood of women with recurrent spontaneous abortions in order to reveal the functional role of this cell population in spontaneous abortions. An extremely high partner allo-antigen-specific cytotoxic T-lymphocyte precursor frequency was determined in the case of all those habitual aborters, where no other than an immunological cause could be responsible for the abortions. This phenomenon supports the important role of the T-lymphocytes in this disorder. We suggest that the immunological background of recurrent spontaneous miscarriages might be determined on the basis of a very high cytotoxic T-lymphocyte precursor frequency. This diagnostic test might be useful in selecting patients for immunotherapy.

Human alloantigen-specific anergic cells induced by a combination of CTLA4-Ig and CsA maintain anti-leukemia and anti-viral cytotoxic responses.

The success of allogeneic hematopoietic stem cell transplantation from HLA-disparate donors depends on the development of new strategies for graft-versus-host disease prevention able to target specifically donor antihost alloreactivity, while preserving GVL and antiviral immune surveillance. Recent experimental and clinical work has shown the feasibility of an approach based on induction of anergy to host alloantigens through blockade of B7/CD28 costimulatory signal in donor T cells, but data on the impact of this strategy on the recovery of the immune system are still lacking. We devised an ex vivo method for induction of host alloantigen-specific unresponsiveness based on treatment with the B7/CD28 blocking agent CTLA4-Ig associated with CsA. We then proceeded to assess the maintenance of an effective immune response towards viral pathogens and tumor cells after CTLA4-Ig/CsA treatment, by measuring the frequency of CTL precursors directed against CMV- and EBV-infected targets, and against autologous leukemic blasts. We demonstrated that (1) CTLA4-Ig and CsA can act synergistically in inducing a state of unresponsiveness to alloantigens; (2) the number of leukemia-reactive, EBV-specific and CMV-specific CTLp is not impaired by CTLA4-Ig/CsA treatment. Our data provide the first direct in vitro evidence that it is possible to preserve antiviral and antileukemia effector cells after blockade of CD28/B7 interaction during MLR.

Immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human IL-2 cDNA: induction of CD8(+) T-cell immunity and NK activity.

Intratumoral (i.t.) injections of an adenovirus encoding the human interleukin-2 (IL-2) under the control of the RSV (Ad-pRSV-IL-2) or CMV (Ad-pCMV-IL-2) promoter were performed in established mastocytoma P815 tumors in B6D2 mice. Both early and long-term survival were found increased in mice treated with Ad-pCMV-IL-2 as compared with those obtained with Ad-pRSV-IL-2: tumor regression was observed in 30--50% of mice for the former and 5--15% for the latter. Difference in efficacy between the two vectors was directly correlated to the amount of IL-2 produced i.t. between 24 and 48 hours postinjection, which reached 10--20 ng/tumor for Ad-pCMV-IL-2 and 0.3--0.5 ng/tumor for Ad-pRSV-IL-2. In both cases, expression in the tumor was clearly detectable for a period of 7--10 days postinjection. Serum IL-2 was not detectable in mice treated with Ad-pRSV-IL-2, whereas expression peaked at a total of 1--2 ng at 24 hours but declined very rapidly in the Ad-pCMV-IL-2-treated group. Constant production of IL-2 inside the tumor was necessary for successful therapy because i.t. injections of recombinant IL-2 at levels up to 1 microg for five consecutive days did not lead to antitumoral activity. Evidence of induced systemic immunity following Ad-pCMV-IL-2 injections was obtained from rechallenge experiments in which tumor-free mice after treatment rejected a subsequent contralateral injection of a lethal dose of P815 tumor cells and from the observation that regression of nontreated tumors occurred in animals bearing bilateral tumors that were treated i.t. in a single tumor with Ad-pCMV-IL-2. P815-specific cytotoxic T lymphocytes (CTL) were found specifically in spleen cells from cured mice or rechallenged mice but not in control mice. Interestingly, limiting dilution analysis of anti-P815 CTL precursor (CTLp) frequency revealed a significant increase in mice cured of their tumor as compared to that obtained in naive mice or control mice treated or not with Ad-IL-2 but whose tumor was growing. In vivo depletion of T-cell subsets, as well as natural killer cells at the time of i.t. injections with Ad-pCMV-IL-2, demonstrated that both CD8(+) T cells and natural killer cells, but not CD4(+) T cells, were required for successful therapy. Finally, mice preimmunized with Ad-null viruses were severely compromised in their capacity to eradicate established P815 tumors after Ad-pCMV-IL-2 therapy, at least when neutralizing antibody titers reached a critical level.

Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65.

Antigen-specific CD8(+) T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-gamma) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8(+) T-cell IFN-gamma expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a (51)Cr release assay and frequencies of peptide-activated CD8(+) T cells expressing IFN-gamma at 6 h (r(2) = 0.72) or 7 days (r(2) = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-gamma expression can be a functional surrogate for identification of CTL precursor cells.

Impaired cytotoxic T lymphocyte response to Epstein-Barr virus-infected NK cells in patients with severe chronic active EBV infection.

Clinical evidence of a relationship between severe chronic active Epstein-Barr virus (EBV) infection and clonal expansion of EBV-infected T or NK cells has been accumulated. In order to clarify pathogenesis of EBV-infected cell proliferation in patients with severe chronic active EBV infection, cytotoxic T lymphocyte (CTL) responses of two patients against B-lymphoblastoid cell lines (B-LCL) and EBV-infected NK cells were examined in comparison with those of HLA-identical healthy siblings. Unexpectedly, patients' CTL activities induced by mixed culture with autologous B-LCLs were markedly reduced, although uncontrolled EBV-related B-cell proliferations have never been experienced. In contrast, limiting dilution analysis demonstrated that B-LCL-specific CTL precursor (CTLp) frequencies of patients were comparable to those of their healthy sisters. The existence of normal levels of B-LCL-specific T cell responses was confirmed by flow-cytometric analysis of IFN-gamma-producing T cells after stimulation with B-LCLs. Infected NK-cell-specific CTLp frequencies of the patients were at undetectable levels despite their expression of latent membrane protein (LMP) 1, suggesting mechanisms to escape immunologic surveillance. In the patients' HLA-identical healthy sisters, infected NK-cell-specific CTLps were detected, and infected NK-cell-specific CTL clones could be established. From these findings, two treatment options for severe chronic active EBV infection are offered for consideration: adoptive transfer of in vitro-cultured CTL, and bone marrow transplantation from HLA-identical donors.

Primary human herpesvirus 8 infection generates a broadly specific CD8+ T-cell response to viral lytic cycle proteins

AbstractHuman herpesvirus 8 (HHV-8) is a recently discovered gammaherpesvirus that is the etiologic agent of Kaposi sarcoma (KS). The natural history of primary HHV-8 infection, including clinical outcome and host immune responses that may be important in preventing disease related to HHV-8, has not been elucidated. The present study characterized the clinical, immunologic, and virologic parameters of primary HHV-8 infection in 5 cases detected during a 15-year longitudinal study of 108 human immunodeficiency virus type 1 seronegative men in the Multicenter AIDS Cohort Study. Primary HHV-8 infection was associated with mild, nonspecific signs and symptoms of diarrhea, fatigue, localized rash, and lymphadenopathy. There were no alterations in numbers of CD4+ or CD8+ T cells or CD8+ T-cell interferon γ (IFN-γ) production to mitogen or nominal antigen. CD8+ cytotoxic T-lymphocyte precursor (CTLp) and IFN-γ reactivity were detected during primary HHV-8 infection, with broad specificity to 5 lytic cycle proteins of HHV-8 encoded by open reading frame 8 (ORF 8; glycoprotein B homolog of Epstein-Barr virus), ORF 22 (gH homolog), ORF 25 (major capsid protein homolog), ORF 26 (a minor capsid protein homolog), or ORF 57 (an early protein homolog), in association with increases in serum antibody titers and appearance of HHV-8 DNA in blood mononuclear cells. CD8+ T-cell responses to HHV-8 decreased by 2 to 3 years after primary infection. This antiviral T-cell response may control initial HHV-8 infection and prevent development of disease.

Suicide induced by cytolytic activity controls the differentiation of memory CD8(+) T lymphocytes.

Cytotoxic T lymphocytes (CTL) confer protection against intracellular pathogens, yet the mechanism by which some escape activation induced cell death (AICD) and give rise to long-lived memory cells is unclear. We studied the differentiation of transgenic TCR CD8(+) cells into CTL and memory cells using a novel system that allowed us to control cytolytic activity. The perforin/granzyme granules used to lyse targets induced the apoptosis of CTL in a fratricide-independent manner. After adoptive transfer to antigen-free mice, the ability of CTL to give generate memory cells was determined. We found that the extent of cytolysis by a common pool of CTL controlled the differentiation into memory cells, which were only generated under conditions of minimal cytolytic activity. Thus, the differentiation of naive CD8(+) cells into memory cells may not depend on the presence on a subset of committed CTL precursors, but rather is controlled by the extent of granule-mediated cytolysis.

Dendritic-cell-peptide immunization provides immunoprotection against bcr-abl-positive leukemia in mice.

Chronic myelogenous leukemia (CML) is a clonal disorder characterized by proliferation of cells that possess the bcr-abl fusion gene resulting in the production of one of two possible chimeric 210-kDa tyrosine kinase proteins. Since these chimeric proteins are expressed only in leukemic cells they have the potential to serve as tumor-specific antigens for cytotoxic T lymphocytes (CTL). Using the 12B1 murine leukemia cell line, derived by retroviral transformation of BALB/c bone marrow cells with the bcr-abl (b3a2) fusion gene, we have demonstrated that intravenous inoculation of 12B1 cells into BALB/c mice results in a disseminated acute leukemia analogous to human CML in blast crisis. Histological sections of liver and spleen and polymerase chain reaction analysis of peripheral blood, bone marrow, liver, spleen and lymph nodes confirmed the presence of bcr-abl+ leukemia cells in these murine tissues, while Western blot data demonstrated the expression of the fusion protein in 12B1 cells. Immunization of mice with dendritic cells (DC) loaded with the synthetic bcr-abl chimeric nonapeptide, GFKQSSKAL, led to a 150 times higher frequency of bcr-abl-specific CTL precursors in the spleen than in mice immunized with peptide alone. In vitro re-stimulation of DC-peptide-primed splenocytes resulted in substantial secretion of interferon gamma and augmented cytolytic activity against 12B1 targets. Finally, vaccination with peptide-loaded DC significantly prolonged survival of BALB/c mice that were challenged with 12B1 leukemia. The capacity to generate bcr-abl-specific CTL in vivo by DC-based immunization may have clinical implications in the treatment of CML.

Expansion of HBV-specific memory CTL primed by dual HIV/HBV genetic immunization during SHIV primary infection in rhesus macaques

We have previously shown the induction of humoral and cytotoxic responses specific for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) antigens, following genetic immunization of rhesus macaques with a plasmid encoding both the third variable domain of the HIV-1 external envelope glycoprotein and the pseudo-viral particle of hepatitis B surface antigen (HBsAg) as presenting molecules. The DNA-immunized primates and two control animals were then challenged with a chimeric simian/human immunodeficiency virus (SHIV). They were all infected. Significant frequencies of SHIV specific cytotoxic T lymphocyte precursors (CTLp) were detected early in peripheral blood. But, in all DNA-immunized macaques, HBV envelope specific CTLp were detected during the primary infection, and they were correlated with the peak of SHIV viremia. Furthermore, HBV or SHIV specific cytotoxicity corresponded in part to CD8 + T cells presenting a memory phenotype. Several mechanisms could account for this cellular response. But our results suggest that an expansion of memory cytotoxic CD8 + cells, not restricted to SHIV specific effectors, could occur in peripheral blood during SHIV primary infection.

In vitro tumor-pulsed or in vivo Flt3 ligand–generated dendritic cells provide protection against acute myelogenous leukemia in nontransplanted or syngeneic bone marrow–transplanted mice

To determine whether immune stimulation could reduce acute myelogenous leukemia (AML) lethality, dendritic cells (DCs) were pulsed with AML antigens and used as vaccines or generated in vivo by Flt3 ligand (Flt3L), a potent stimulator of DC and natural killer (NK) cell generation. Mice were then challenged with AML cells. The total number of splenic anti-AML cytotoxic T-lymphocyte precursors (CTLPs) present at the time of challenge was increased 1.9-fold and 16.4-fold by Flt3L or DC tumor vaccines, respectively. As compared with the 0% survival of controls, 63% or more of recipients of pulsed DCs or Flt3L survived long term. Mice given AML cells prior to DC vaccines or Flt3L had only a slight survival advantage versus non-treated controls. NK cells or NK cells and T cells were found to be involved in the antitumor responses of Flt3L or DCs, respectively. DC vaccines lead to long-term memory responses but Flt3L does not. Syngeneic bone marrow transplantation (BMT) recipients were analyzed beginning 2 months post-BMT. In contrast to the uniform lethality in BMT controls given AML cells, recipients of either Flt3L or DC vaccines had a significant increase in survival. The total number of splenic anti-AML CTLPs at the time of AML challenge in BMT controls was 40% of concurrently analyzed non-BMT controls. Flt3L or DC vaccines increased the total anti-AML CTLPs 1.4-fold and 6.8-fold, respectively. Neither approach was successful when initiated after AML challenge. It was concluded that DC vaccines and Flt3L administration can enhance an AML response in non-transplanted or syngeneic BMT mice but only when initiated prior to AML progression.

Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus.

IFN-gamma-deficient (IFN-gamma(-/-)) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4(+)-depleted mice, especially in mice also deficient in IFN-gamma. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-gamma(-/-) mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-gamma(-/-) hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8(+) effector T cells is one important factor influencing the requirement for T cell help during viral infection.

Viral IL-10-induced immunosuppression requires Th2 cytokines and impairs APC function within the allograft.

Previous reports demonstrated that retroviral mediated gene transfer of viral IL-10 (vIL-10) prolongs allograft survival by decreasing donor-specific cytotoxic T lymphocyte precursor (CTLp) and IL-2-secreting helper T lymphocyte precursor (HTLp) frequency within graft-infiltrating cells (GIC). This report now shows that vIL-10 efficacy is dependent on CD4(+) T cells, suggesting that immunosuppression may involve a switch from a Th1 to a Th2 alloresponse. In support of this, anti-IL-4 or anti-murine IL-10 (anti-mIL-10) mAbs, but not anti-IFN-gamma mAb, administered at the time of vIL-10 gene transfer prevents enhanced graft survival. Because Th switching involves APC function, GIC were examined for their ability to present alloantigen. GIC from vIL-10-treated grafts were shown to be mostly of recipient (CBA) origin, yet were unable to elicit alloproliferative responses from donor type (C57BL/6) or third party (BALB/c) responders. The inability of vIL-10-treated GIC to stimulate the MLR was not due to the generation of negative regulatory cells or the production of immunosuppressive cytokines such as IL-4, mIL-10, or TGFbeta. Using fractionated GIC subpopulations, the number of recipient type cells in the allografts was modestly reduced by vIL-10 gene transfer, while maintaining both APC phenotype and alloantigen presenting function. Conversely, after vIL-10 gene transfer, donor type GIC were unable to participate in direct alloantigen presentation. Therefore, local immunosuppression induced by vIL-10 gene transfer is CD4 T cell and IL-4 and mIL-10 dependent, and impairs direct alloantigen presentation through an alteration of donor type APC function.

In vitro HIV-specific CTL activity from HIV-seropositive individuals is augmented by interleukin-12 (IL-12).

IL-12 production is reduced in HIV infection, and recombinant human IL-12 (rhIL-12) augments in vitro HIV-specific proliferative responses in PBMC from HIV-seropositive individuals. To determine whether rhIL12 could also augment HIV-specific CTL responses we studied 41 HIV-seropositive individuals. Recombinant hIL-12 increased the detectable in vitro HIV-specific CD8 CTL activity of PBMC taken from HIV-seropositive individuals with CD4 counts >500 cells/microl and from some individuals with lower CD4 counts. IL-12 increased cell recovery in cultures of PBMC from HIV-seropositive individuals with CD4 counts >500 cells/microl and also increased the precursor CTL frequency. However, the increase in HIV-specific CTL activity was not due to IL-2 or IFN-gamma production or an increase in the number of cells with surface markers characteristic of CTL effector cells. This study demonstrates that rhIL-12 augments in vitro HIV-specific CTL activity and provides evidence to justify further investigation within clinical trials of this cytokine in HIV infection.

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR+ cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24+ CAR+ cancer patients. In addition, the CTL responses were obtained from three of ten CAR– cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR+ cancer patients and that of CAR– cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR+ cancer patients. Moreover, in cases other than CAR+ cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24+ cancer patients with a recoverin-expressing tumor.